Back to EveryPatent.com
United States Patent |
5,747,343
|
Tsuchiya
,   et al.
|
May 5, 1998
|
Leukocyte classification reagent
Abstract
A leukocyte classification reagent comprising at least one surface active
agent selected from the group consisting of anionic active agents and
amphoteric active agents lyses erythrocytes within a short period of time
when added to a blood sample but does not cause damage on leukocytes for a
certain period of time keeping their original state or close to the
original state so that leukocytes can be classified directly into four
groups, namely lymphocyte, monocyte, neutrophil and eosinophil, by
measuring forward scattering and side-way scattering by a flow cytometer
after lysis of erythrocytes.
Inventors:
|
Tsuchiya; Katsuhiro (Tokyo, JP);
Nagai; Yutaka (Tokyo, JP);
Ikeda; Mami (Tokyo, JP)
|
Assignee:
|
Nihon Kohden Corporation (Tokyo, JP)
|
Appl. No.:
|
550861 |
Filed:
|
October 31, 1995 |
Foreign Application Priority Data
Current U.S. Class: |
436/63; 436/10; 436/17; 436/18 |
Intern'l Class: |
G01N 033/48 |
Field of Search: |
436/10,17,18,63
|
References Cited
U.S. Patent Documents
4528274 | Jul., 1985 | Carter et al. | 436/10.
|
4637986 | Jan., 1987 | Brown et al. | 436/10.
|
5116539 | May., 1992 | Hamaguchi et al. | 252/408.
|
5128265 | Jul., 1992 | Meiattini | 436/17.
|
5242832 | Sep., 1993 | Sakata et al. | 436/17.
|
5250437 | Oct., 1993 | Toda et al. | 436/10.
|
5264369 | Nov., 1993 | Sakata et al. | 436/63.
|
5389549 | Feb., 1995 | Hamaguchi et al. | 436/10.
|
5413938 | May., 1995 | Tsujino et al. | 436/63.
|
5496734 | Mar., 1996 | Sakata | 436/63.
|
5538893 | Jul., 1996 | Sakata et al. | 436/10.
|
Foreign Patent Documents |
0214613 | Mar., 1987 | EP.
| |
0316453A1 | May., 1988 | EP.
| |
0424871 | May., 1991 | EP.
| |
Primary Examiner: Warden; Jill
Assistant Examiner: Carrillo; Sharidan
Attorney, Agent or Firm: Sughrue, Mion, Zinn, Macpeak & Seas, PLLC
Claims
What is claimed is:
1. A leukocyte classification reagent which consists essentially of at
least one surface active agent selected from the group consisting of an
anionic active agent and an amphoteric active agent in an amount effective
to classify leukocytes by lysing erythrocytes and acting upon leukocytes,
wherein said anionic active agent is an alkyl sulfate anionic active agent
represented by the formula (1):
R--O--SO.sub.3 X (1)
wherein R represents an alkyl or alkylene group having 8 to 18 carbon atoms
and X represents Na, K, NH.sub.4 or HN(CH.sub.2 CH.sub.2 OH).sub.3.
2. The reagent according to claim 1, wherein said alkyl sulfate anionic
active agent is sodium lauryl sulfate.
3. A leukocyte classification reagent which comprises at least one surface
active agent selected from the group consisting of an anionic active agent
and an amphoteric active agents in an amount effective to classify
leukocytes by lysing erythrocytes and acting upon leukocytes,
wherein said anionic active agent is a polyoxyethylene anionic active agent
represented by the formula (2):
R--O--(CH.sub.2 CH.sub.2 O).sub.n --SO.sub.3 --X (2)
wherein R represents an alkyl or alkylene group having 8 to 18 carbon
atoms, n is an integer of 1 to 5 and X represents Na, K, NH.sub.4 or
HN(CH.sub.2 CH.sub.2 OH).sub.3.
4. The reagent according to claim 3, wherein said anionic active agent is
selected from the group consisting of sodium polyoxyethylene (3)
C.sub.12-13 alkyl ether sulfate, sodium polyoxyethylene (3) C.sub.11-15
alkyl ether sulfate, and polyoxyethylene (3) C.sub.11-15 alkyl ether
triethanolamine sulfate.
5. A leukocyte classification reagent which consists essentially of at
least one surface active agent selected from the group consisting of an
anionic active agent and an amphoteric active agent in an amount effective
to classify leukocytes by lysing erythrocytes and acting upon leukocytes,
wherein said anionic active agent is a coconut fatty acid anionic active
agent represented by the formula (3):
##STR5##
wherein R represents an alkyl or alkylene group having 8 to 18 carbon
atoms, X represents CH.sub.2 SO.sub.3 or COO and Y represents Na, K,
NH.sub.4 or HN(CH.sub.2 CH.sub.2 OH).sub.3.
6. The reagent according to claim 5 wherein said coconut fatty acid anionic
active agent is sodium coconut fatty acid methyltaurine or sodium coconut
fatty acid sarcosine.
7. A leukocyte classification reagent which comprises at least one surface
active agent selected from the group consisting of an anionic active agent
and an amphoteric active agents in an amount effective to classify
leukocytes by lysing erythrocytes and acting upon leukocytes,
wherein said amphoteric active agent is a
2-alkyl-N-carboxymethyl-N-hydroxyethyl imidazoliniumbetaine represented by
the formula (4):
##STR6##
wherein R represents an alkyl or alkylene group having 6 to 21 carbon
atoms.
8. A leukocyte classification reagent which comprises at least one surface
active agent selected from the group consisting of an anionic active agent
and an amphoteric active agents in an amount effective to classify
leukocytes by lysing erythrocytes and acting upon leukocytes,
wherein said amphoteric active agent is 2-alkyl(C.sub.8 H.sub.17 -C.sub.18
H.sub.37)-N-carboxymethyl-N-hydroxyethyl imidazoliniumbetaine.
9. A process for classifying of leukocytes which comprises the steps of:
(A) adding a leukocyte classification reagent to a blood sample solution to
lyse erythrocytes present in said blood sample solution and producing a
hemolysate; and
(B) subjecting the hemolysate of step (A) to flow cytometry to measure
forward optical scattering and side-way optical scattering thereby
producing a distribution diagram which classifies the leukocytes into a
plurality of groups,
wherein said reagent consists essentially of at least one surface active
agent selected from the group consisting of an alkyl sulfate anionic
active agent and an amphoteric active agent in an amount effective to
classify leukocytes by lysing erythrocytes and acting upon leukocytes,
wherein said alkyl sulfate anion active agent is represented by the
formula (1):
R--O--SO.sub.3 X (1)
wherein R represents an alkyl or alkylene group having 8 to 18 carbon atoms
and X represents Na, K, NH.sub.4 or HN(CH.sub.2 CH.sub.2 OH).sub.3.
10. The process according to claim 9, wherein said alkyl sulfate anionic
active agent is sodium lauryl sulfate.
11. A process for classifying of leukocytes which comprises the steps of:
(A) adding a leukocyte classification reagent to a blood sample solution to
lyse erythrocytes present in said blood sample solution and producing a
hemolysate; and
(B) subjecting the hemolysate of step (A) to flow cytometry to measure
forward optical scattering and side-way optical scattering thereby
producing a distribution diagram which classifies the leukocytes into a
plurality of groups,
wherein said reagent consists essentially of at least one surface agent
selected from the group consisting of a coconut fatty acid anionic active
agent and an amphoteric active agent in an amount effective to classify
leukocytes by lysing erythrocytes and acting upon leukocytes, wherein said
coconut fatty acid anionic active agent is represented by the formula (3):
##STR7##
wherein R represents an alkyl or alkylene group having 8 to 18 carbon
atoms, X represents CH.sub.2 SO.sub.3 or COO and Y represents Na, K,
NH.sub.4 or HN(CH.sub.2 CH.sub.2 OH).sub.3.
12. The process according to claim 11, wherein said coconut fatty acid
anionic active agent is sodium coconut fatty acid methyltaurine or sodium
coconut fatty acid sarcosine.
13. The reagent according to claims 4, 3, 7 or 8, wherein said reagent has
a pH value ranging from 3 to 11.
14. The reagent according to claims 4, 3, 7 or 8, wherein said reagent has
an osmotic pressure of from 250 to 400 mOsm/kgH.sub.2 O.
15. A process for classifying of leukocytes which comprises:
(A) adding the leukocyte classification reagent of claims 4, 3, 7 or 8, to
a blood sample solution to lyse erythrocytes present in said blood sample
solution and producing a hemolysate; and
(B) subjecting the hemolysate of step (A) to flow cytometry to measure
forward scattering and side-way scattering thereby producing a
distribution diagram which classifies the leukocytes into a plurality of
groups.
16. The reagent according to claim 1, 6, 2 or 5, wherein said reagent has a
pH value ranging from 3 to 11.
17. The reagent according to claim 1, 6, 2 or 5, wherein said reagent has
an osmotic pressure of from 250 to 400 mOsm/kgH.sub.2 O.
18. The process according to claim 9, 10, 11 or 12, wherein said reagent
has a pH value ranging from 3 to 11.
19. The process according to claim 9, 10, 11 or 12, wherein said reagent
has an osmotic pressure of from 250 to 400 mOsm/kgH.sub.2 O.
Description
FIELD OF THE INVENTION
This invention relates to a reagent to be used in the field of clinical
inspection for the classification of blood cells, more particularly to a
reagent for use in a method for the classification of leukocytes in which
blood cells after lysis of erythrocytes are optically measured using a
flow cytometer.
BACKGROUND OF THE INVENTION
In a commonly used method for the classification and counting of
leukocytes, an erythrocyte lysing agent (to be referred to as "hemolytic
agent" hereinafter) mainly composed of a quaternary ammonium salt as a
cationic surface active agent is added to a diluted blood solution to
dissolve membranes and cytoplasmic contents of erythrocytes and
leukocytes, and the remaining leukocyte nuclei are measured based on the
Coulter's theory to count the number of leukocytes. Since leukocytes are
observed in the shrunken form, this method has a problem in that
information for the classification of leukocytes cannot be obtained.
In a method which has been developed with the aim of overcoming this
problem by improving components of the hemolytic agent and their
concentrations and slackening the reaction of leukocytes, leukocytes are
classified into three groups, namely lymphocyte, monocyte and granulocyte,
based on the difference in their lysing rate and electric conductivity.
However, great concern has been directed in recent years toward the
development of a method which can classify leukocytes into five groups
including lymphocyte, monocyte, neutrophil, eosinophil and basophil, or at
least four groups excluding basophil, because such a method can contribute
to the diagnosis of diseases by counting blood leukocytes as individual
groups.
On the other hand, a flow cytometer has been used for the identification
and analysis of cells and fine particles based on light scattering. As
shown in FIG. 10, a flow analyzer 4 of this cell measuring apparatus (flow
cytometer) is designed to measure scattering from sample particles. That
is, a sample solution containing particles to be measured is introduced
into the flow analyzer 4 where laser beams generated from a laser source 1
are irradiated through radiation scattering lenses 2 and 3, the forward
scattering after stoppage of direct light through an irradiation light
stopper is measured by a forward scattering detector 9 via a forward
scattering detection lens 8, and the voltage level measured by the
detector is recorded in the analyzer 10. On the other hand, the side-way
scattering in the flow analyzer 4 is measured by a side-way scattering
detector 6 via a side-way scattering detection lens 5, and the voltage
level measured by the detector is recorded in the analyzer 10. Based on
both of the voltage levels in the analyzer 10, a two-dimensional
distribution diagram (scattergram) of the forward scattering and side-way
scattering is displayed on a display device 11.
However, such a measurement by a flow cytometer can count the number of
leukocytes but it can hardly classify them even into three groups, because
leukocyte membranes are lysed in significant quantity when the
above-described hemolytic agent mainly composed of a quaternary ammonium
salt surface active agent is used.
In a method in which leukocytes are classified by measuring forward
scattering and side-way scattering making use of a flow cytometer,
leukocytes are classified into three groups, namely lymphocyte, monocyte
and granulocyte, by adding a hemolytic agent containing ammonium oxalate
as its main component to a diluted blood solution to effect selective
lysis of erythrocytes. This method, however, requires 20 to 30 minutes for
the lysis of erythrocytes so that it cannot be used as a method for use in
the treatment of a large number of samples. What is more, since
distribution of each leukocyte group is not clear and eosinophil is
included in the distribution of neutrophil, this method cannot isolate
eosinophil as its single distribution.
For the purpose of overcoming the above-described problems, a leukocyte
classification agent has been proposed (cf. WO 88/09504) which contains at
least one surface active agent (hereinafter sometimes referred to as
merely "active agent") selected from the group consisting of
polyoxyethylene anionic active agents and polyoxyethylene nonionic active
agents and which can classify leukocytes into 3 to 5 groups.
Both of these polyoxyethylene anionic and nonionic active agents are
commonly represented the formula
R.sub.1 --R.sub.2 --(CH.sub.2 CH.sub.2 O).sub.n --A
wherein R.sub.1 represents an alkyl or alkenyl group having 10 to 22 carbon
atoms, R.sub.2 represents O,
##STR1##
or COO, n is an integer of from 8 to 30 and A represents SO.sub.3 Na,
COONa, OSO.sub.3 Na or ONa in the case of the anionic active agent or H in
the case of the nonionic active agent.
Thus, it has a relatively large ethylene oxide addition mol number of from
8 to 30. Such a large ethylene oxide addition mol number causes reduction
of cytolytic activity. In addition, when a two-dimensional distribution
diagram is obtained based on the results of measurements by RF and DC
methods making use of this reagent, separation of lymphocyte, monocyte and
granulocyte is poor and eosinophil cannot be measured at the same time.
SUMMARY OF THE INVENTION
In view of the above, an object of the present invention is to provide a
leukocyte classification reagent (hemolytic agent) which lyses
erythrocytes within a short period of time when added to a blood sample
solution but does not cause damage on leukocytes for a certain period of
time keeping their original state or close to the original state, so that
leukocytes can be classified directly into four groups, namely lymphocyte,
monocyte, eosinophil and neutrophil, by measuring forward scattering and
side-way scattering by a flow cytometer after lysis of erythrocytes.
Accordingly, the present invention provides a leukocyte classification
reagent which comprises at least one surface active agent selected from
the group consisting of anionic active agents and amphoteric active agents
in an amount effective to classify leukocytes in a blood sample by lysing
erythrocytes and acting upon leukocytes. Specifically, the above anionic
active agent is an alkyl sulfate anionic active agent represented by the
formula (1):
R--O--SO.sub.3 X (1)
wherein R represents an alkyl or alkylene group having 8 to 18 carbon atoms
and X represents Na, K, NH.sub.4 or HN(CH.sub.2 CH.sub.2 OH).sub.3 ; or a
polyoxyethylene anionic active agent represented by the formula (2):
R--O--(CH.sub.2 CH.sub.2 O).sub.n --SO.sub.3 X (2)
wherein R represents an alkyl or alkylene group having 8 to 18 carbon
atoms, n is an integer of 1 to 5 and X represents Na, K, NH.sub.4 or
HN(CH.sub.2 CH.sub.2 OH).sub.3. More specifically, the polyoxyethylene
anionic active agent is selected from the group consisting of sodium
polyoxyethylene(3) C.sub.12-13 alkyl ether sulfate, sodium
polyoxyethylene(3) C.sub.11-15 alkyl ether sulfate, polyoxyethylene(3)
alkyl C.sub.11-15 alkyl ether triethanolamine and sodium lauryl sulfate.
The anionic active agent can also be a coconut fatty acid anionic active
agent represented by the formula (3):
##STR2##
wherein R represents an alkyl or alkylene group having 8 to 18 carbon
atoms, X represents CH.sub.2 SO.sub.3 or COO and Y represents Na, K,
NH.sub.4 or HN(CH.sub.2 CH.sub.2 OH).sub.3. Specifically, the coconut
fatty acid anionic active agent is sodium coconut fatty acid methyltaurine
or sodium coconut fatty acid sarcosine.
The above-described amphoteric active agent is a
2-alkyl-N-carboxymethyl-N-hydroxyethyl imidazoliniumbetaine represented by
the formula (4):
##STR3##
wherein R represents an alkyl or alkylene group having 6 to 21 carbon
atoms. Specifically, the amphoteric active agent is 2-alkyl(C.sub.8
H.sub.17 -C.sub.18 H.sub.37)-N-carboxymethyl-N-hydroxyethyl
imidazoliniumbetaine.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a two-dimensional distribution diagram when sodium
polyoxyethylene(3) C.sub.12-13 alkyl ether sulfate was used as a hemolytic
agent.
FIG. 2 is a two-dimensional distribution diagram when sodium
polyoxyethylene(3) C.sub.11-15 alkyl ether sulfate was used as a hemolytic
agent.
FIG. 3 is a two-dimensional distribution diagram when sodium lauryl sulfate
was used as a hemolytic agent.
FIG. 4 is a two-dimensional distribution diagram when sodium lauryl sulfate
was used as a hemolytic agent and classification of leukocytes was carried
out at pH 10.
FIG. 5 is a two-dimensional distribution diagram when sodium lauryl sulfate
was used as a hemolytic agent and classification of leukocytes was carried
out at pH 3.
FIG. 6 is a two-dimensional distribution diagram when sodium
polyoxyethylene(3) C.sub.11-15 alkyl ether sulfate was used as a hemolytic
agent and classification of leukocytes was carried out under an osmotic
pressure of 450 mOsm/kg H.sub.2 O.
FIG. 7 is a two-dimensional distribution diagram when sodium
polyoxyethylene(3) C.sub.11-15 alkyl ether sulfate was used as a hemolytic
agent and classification of leukocytes was carried out under an osmotic
pressure of 220 mOsm/kg H.sub.2 O.
FIG. 8 is a two-dimensional distribution diagram when sodium coconut fatty
acid methyltaurine was used as a hemolytic agent.
FIG. 9 is a two-dimensional distribution diagram when 2-C.sub.8-18
alkyl-N-carboxymethyl-N-hydroxyethyl imidazoliniumbetaine was used as a
hemolytic agent.
In the above drawings, 0 represents erythrocyte ghost, 1 represents
lymphocyte, 2 represents monocyte, 3 represents neutrophil and 4
represents eosinophil.
FIG. 10 is a schematic illustration showing the optical system of a flow
cytometer in which the leukocyte classification reagent of the present
invention is used.
DETAILED DESCRIPTION OF THE INVENTION
Any anionic and amphoteric surface active agent can be used in the
leukocyte classification reagent of the present invention as long as it
enables lysis of erythrocytes within a short period of time when they are
added to a blood sample solution without causing damage on leukocytes to
maintain the original state of leukocytes or close to the original state.
In the alkyl sulfate anionic active agent represented by the formula (1),
the group R preferably has 8 to 18 carbon atoms. In the above-described
polyoxyethylene anionic active agent represented by the formula (2), R
preferably has 8 to 18 carbon atoms and ethylene oxide addition mol
numbers of 1 to 5. Hydrogen in the alkyl or alkylene group R takes a great
role in the lysis of cells. The longer the chain length of R is, the
stronger the cytolytic activity is, while the shorter the chain length is,
the weaker the cytolytic activity is. However, the water solubility of the
surface active agent becomes lower as the chain length of R becomes longer
and the agent is sparingly soluble or makes a hemolysate turbid. The
number of carbon atoms in the group R within the above-described range
balances these cases with each other. Also, as the integer represented by
n becomes larger, hydrophilicity of the agent increases, but its cytolytic
activity is reduced. The group X does not take particular role in the
reaction upon blood cells.
The above description concerning the carbon number of R is also applicable
to the coconut fatty acid anionic active agent represented by the formula
(3), and thus it preferably has 8 to 18 carbon atoms. In the same manner
as described above, both of the groups X and Y in the formula (3) do not
take particular role in the reaction upon blood cells.
The amphoteric active agent acts as an anionic active agent or a cationic
active agent depending on the pH range, and it acts as an anionic active
agent when used as a hemolytic agent at a pH value of 7 or higher. In the
betaine amphoteric active agent represented by the formula (4), R
preferably has 6 to 21 carbon atoms.
According to the present invention, each blood sample is diluted with a
usual diluent in order to effect uniform dispersion of blood, prior to the
addition of a hemolytic agent as the leukocyte classification reagent.
Such a diluent comprises an osmotic pressure adjusting agent, a buffer
agent, a chelating agent and an antimicrobial agent dissolved in proper
amounts in distilled water, with its typical composition shown below.
Diluent
______________________________________
sodium chloride 9.0 g
potassium dihydrogenphosphate
0.91 g
disodium hydrogenphosphate
9.55 g
EDTA 2Na 0.3 g
sodium 1-hydroxypyridine-2-thionate
0.25 g
distilled water 1000 ml
______________________________________
The osmotic pressure adjusting agent is used to adjust the osmotic pressure
of blood sample solutions to such a level that blood cells can exist
stably, namely 250 to 400 mOsm/kg H.sub.2 O. When the osmotic pressure is
higher than this range, hemolytic activity becomes strong, particularly
accelerating lysis of erythrocytes, and neutrophils shrink a little. On
the other hand, when the osmotic pressure is lower than this range,
hemolytic activity becomes weak and the amount of erythrocyte ghost
(unlysed erythrocyte residue) becomes particularly large.
It is desirable that the buffer agent can maintain blood sample solutions
within a pH range of approximately from 6.8 to 7.6 so that blood cells can
exist stably. However, when a hemolytic agent is added to a blood sample
solution, the pH value must be within the optimum range of the agent. In
consequence, a pH value outside the above range is applicable with the
proviso that it does not spoil the purpose of the present invention.
The purpose of the present invention can also be achieved by adding the
hemolytic agent to the diluent in advance or by adding an appropriate
amount of the surface active agent to the diluent directly without
dilution.
The anionic active agent of the present invention acts as the hemolytic
agent generally within the pH range of from 3 to 11, and the amphoteric
active agent within the pH range of from 7 to 11, though the optimum range
varies depending on their kinds. When the pH value is higher than these
ranges, hemolytic activity becomes strong to cause lysis of not only
erythrocytes but also leukocytes. On the other hand, when the pH value is
lower than these ranges, hemolytic activity becomes weak so that
separation of distributions of respective leukocyte components becomes
poor.
The chelating agent is used to effect formation of chelate compounds with
metal ions, in order to prevent generation of precipitate caused by metal
ions during preservation of the diluent for a prolonged period of time or
at the time of its use.
The antimicrobial agent is used to prevent growth of fungi and bacteria
during long-term preservation of the diluent.
The amount of the hemolytic agent which contains the above-described
surface active agent as the main component to be added to a blood sample
diluted with the above-described diluent varies depending on the kind of
the surface active agent. In general, however, the amount of ghost
increases when the concentration of the surface active agent in the blood
sample solution is so low that entire erythrocytes cannot be lysed. On the
other hand, not only erythrocytes but also leukocytes are lysed when the
active agent concentration is high. In addition, the time for hemolysis
varies depending on the added amount of the hemolytic agent. The time for
hemolysis becomes short when the added amount is large, but stable period
of leukocytes also becomes short. In consequence, it is necessary to
select proper amount of the surface active agent to be added. One of
ordinally skilled in the art can easily determine the appropriate
concentration of the surface active agent. For example, 1 ml of a
hemolytic agent prepared by dissolving the polyoxyethylene anionic active
agent represented by the above-described formula (2) in distilled water to
give a concentration of 0.05% can be added to a blood sample obtained by
diluting 50 .mu.l of whole blood with 2 ml of the above-described diluent.
The amount of the hemolytic agent should be determined depending on a
dilution ratio of blood and the concentration of the surface active agent.
Examples of the present invention are given below by way of illustration
and not by way of limitation.
EXAMPLE 1
A polyoxyethylene anionic active agent, sodium polyoxyethylene(3)
alkyl(C.sub.12-13 mixture) ether sulfate (C.sub.12-13 --O--(CH.sub.2
CH.sub.2 O).sub.3 --SO.sub.3 Na), was used in this example as a hemolytic
agent component.
A 1.75 g portion of this anionic active agent was dissolved in 1,000 ml of
distilled water to serve as a hemolytic agent (if necessary, an
appropriate amount of sodium chloride can be added to distilled water for
the adjustment of osmotic pressure).
A 25 .mu.l portion of a blood sample was diluted with 1 ml of a diluent
having the following composition.
______________________________________
sodium chloride 9.0 g
potassium dihydrogenphosphate
0.91 g
disodium hydrogenphosphate
9.55 g
EDTA 2Na 0.3 g
sodium 1-hydroxypyridine-2-thionate
0.25 g
distilled water 1000 ml
______________________________________
Then, 125 .mu.l of the above hemolytic agent was added thereto. Ten seconds
thereafter, strengths of forward scattering and side-way scattering were
measured using the flow cytometer to obtain a two-dimensional distribution
diagram (scattergram) of FIG. 1.
As shown in FIG. 1, leukocytes were clearly classified into four groups,
namely lymphocyte (1), monocyte (2), neutrophil (3) and eosinophil (4). In
this drawing, (0) represents erythrocyte ghost.
In addition, two-dimensional distribution diagrams similar to the diagram
shown in FIG. 1 were obtained when the above-described hemolytic agent
(concentration, 0.175%) was added to the above-described diluted blood
solution within the range of from 100 to 1,000 .mu.l. In this instance,
the purpose of the present invention can also be achieved by adding the
hemolytic agent to the diluent in advance or by adding an appropriate
amount of the above-described surface active agent to the diluent directly
without dilution.
The surface active agent of this example showed the same result shown in
FIG. 1 within the pH range of from 3 to 10.
With regard to the osmotic pressure, the same result shown in FIG. 1 was
also obtained within the range of from 250 to 400 mOsm/kg H.sub.2 O.
EXAMPLE 2
Classification of leukocytes was carried out in the same manner as
described in Example 1, except that a polyoxyethylene anionic active
agent, sodium polyoxyethylene(3) alkyl(C.sub.11-15 mixture) ether sulfate,
was used as a hemolytic agent component and 2.5 g of this anionic active
agent was dissolved in 1,000 ml of distilled water to be used as a
hemolytic agent, with the results shown in FIG. 2. As the result,
leukocytes were clearly classified into four groups similar to the case of
Example 1. Also, the same results shown in FIG. 2 were obtained within the
addition range of from 50 to 300 .mu.l (in the case of 0.25% hemolytic
agent), within the pH range of from 6 to 10 and within the osmotic
pressure range of from 250 to 400 mOsm/kg H.sub.2 O similar to the case of
Example 1.
EXAMPLE 3
Classification of leukocytes was carried out in the same manner as
described in Example 1, except that a polyoxyethylene anionic active
agent, sodium lauryl sulfate (C.sub.12 H.sub.25 OSO.sub.3 Na), was used as
the active ingredient of hemolytic agent and 5.0 g of this anionic active
agent was dissolved in 1,000 ml of distilled water to be used as a
hemolytic agent, with the results shown in FIG. 3. As the result,
leukocytes were clearly classified into four groups similar to the case of
Example 1. Also, in the case of 0.5% hemolytic agent, the same results
shown in FIG. 3 were obtained within the addition range of from 50 to 200
.mu.l, within the pH range of from 5 to 8 and within the osmotic pressure
range of from 250 to 400 mOsm/kg H.sub.2 O similar to the case of Example
1.
EXAMPLE 4
Using two types of the polyoxyethylene anionic active agent represented by
the above-described formula (2) in which R has 8 and 18 carbon atoms and n
is 3 in both types, leukocyte classification tests were carried out in the
same manner as described in Examples 1 to 3. In both cases, scattergrams
similar to those shown in FIGS. 1 to 3 were obtained in which leukocytes
were clearly classified into four groups, namely lymphocyte, monocyte,
neutrophil and eosinophil.
Comparative Example 1
Using sodium lauryl sulfate as the active ingredient of hemolytic agent
similar to the case of Example 3, effects of pH values 10 and 3 on the
classification of leukocytes were examined. The results are shown in FIGS.
4 and 5, respectively. As is evident from the results shown in FIG. 4,
only a hardly measurable two-dimensional distribution diagram is obtained
at pH 10, because the hemolytic activity becomes too strong so that
leukocytes and erythrocytes are lysed at the same time. On the other hand,
as shown in FIG. 5, the hemolytic activity became too weak at pH 3 so that
satisfactory results were not obtained due to poor separation of
leukocytes.
Comparative Example 2
Using sodium polyoxyethylene(3) alkyl(C.sub.11-15 mixture) ether sulfate as
the active ingredient of hemolytic agent similar to the case of Example 2,
effects of the osmotic pressures of 450 and 220 mOsm/kg H.sub.2 O on the
classification of leukocytes were examined. At an osmotic pressures of 450
mOsm/kg H.sub.2 O, the hemolytic activity became so strong that lysis of
erythrocytes progressed especially rapidly but lysis of leukocytes also
progressed and neutrophils shrank a little, so that only a hardly
measurable two-dimensional distribution diagram was obtained as shown in
FIG. 6 similar to the case of high pH value shown in FIG. 4. At an osmotic
pressures of 220 mOsm/kg H.sub.2 O, on the other hand, the hemolytic
activity became so weak that the amount of erythrocyte ghost became
particularly large and the results therefore were not satisfactory as
shown in FIG. 7.
The same results were obtained when the alkyl group R and the ethylene
oxide mol number n are outside the ranges defined in the foregoing.
EXAMPLE 5
A coconut fatty acid anionic active agent, sodium coconut fatty acid
methyltaurine (R-CON(CH.sub.3)CH.sub.2 CH.sub.2 SO.sub.3 Na), was used as
the active ingredient of hemolytic agent.
A 5.8 g portion of this anionic active agent was dissolved in 1,000 ml of
distilled water to serve as a hemolytic agent (if necessary, an
appropriate amount of sodium chloride can be added to distilled water for
the adjustment of osmotic pressure similar to the case of Example 1).
By repeating the procedure of Example 1, a two-dimensional distribution
diagram shown in FIG. 8 was obtained.
As the result, leukocytes were clearly classified into four groups, namely
lymphocyte (1), monocyte (2), neutrophil (3) and eosinophil (4). In this
drawing, (0) represents erythrocyte ghost.
Similar to the case of Example 1, when the active agent was used in a
concentration of 0.58%, the same results shown in FIG. 9 were obtained
within the addition range of from 50 to 300 .mu.l, within the pH range of
from 6 to 9 and within the osmotic pressure range of from 250 to 400
mOsm/kg H.sub.2 O.
EXAMPLE 6
A betaine amphoteric active agent, 2-alkyl(C.sub.8 H.sub.17 -C.sub.18
H.sub.37 mixture)-N-carboxymethyl-N-hydroxyethyl imidazoliniumbetaine
represented by the formula:
##STR4##
was used as the active ingredient of hemolytic agent.
A 3.6 g portion of this amphoteric active agent was dissolved in 1,000 ml
of distilled water to serve as a hemolytic agent.
By repeating the procedure of Example 5, a two-dimensional distribution
diagram shown in FIG. 9 was obtained.
As the result, leukocytes were clearly classified into four groups similar
to the case of Examples 1 to 5.
Also similar to the case of Example 5, when the active agent was used in a
concentration of 0.36%, the same results shown in FIG. 9 were obtained
within the addition range of from 75 to 200 .mu.l, within the pH range of
from 7 to 11 and within the osmotic pressure range of from 250 to 400
mOsm/kg H.sub.2 O.
EXAMPLE 7
Using two types of the betaine amphoteric active agent represented by the
above-described formula (3) in which R has 6 or 21 carbon atoms, leukocyte
classification tests were carried out in the same manner as described in
Example 6. In both cases, two-dimensional distribution diagrams similar to
those shown in FIG. 9 were obtained in which leukocytes were clearly
classified into four groups, namely lymphocyte, monocyte, neutrophil and
eosinophil.
Thus, according to the present invention, the inventive leukocyte
classification reagent can lyse erythrocytes within a short period of time
when added to a diluted blood solution but does not cause damage on
leukocytes, so that the original state of leukocytes or a state close
thereto can be maintained for a certain period of time, and leukocytes
therefore can be classified directly into four groups by simple procedure
and construction, thus exerting great effects in the field of clinical
inspection.
While the invention has been described in detail and with reference to
specific examples thereof, it will be apparent to one skilled in the art
that various changes and modifications can be made therein without
departing from the spirit and scope thereof.
Top